Therapeutic agent for keratoconjunctive disorders

ABSTRACT

The present invention addresses the problem of providing a novel therapeutic agent for keratoconjunctive disorders. As a means for solving the problem, a therapeutic agent for keratoconjunctive disorders which contains a RARγ agonist as an active ingredient is provided. The therapeutic agent exhibits an excellent ameliorating effect in a keratoconjunctive disorder model, and is therefore useful as a therapeutic agent for keratoconjunctive disorders such as corneal ulcer, corneal epithelial abrasion, keratitis, dry eye, conjunctivitis, chronic superficial keratitis, corneal erosion, persistent corneal disorders, superficial punctate keratopathy, corneal epithelial defects, conjunctival epithelial defects, keratoconjunctivitis sicca, superior limbic keratoconjunctivitis, filamentary keratoconjunctivitis, infectious keratitis, noninfectious keratitis, infectious conjunctivitis and noninfectious conjunctivitis. The therapeutic agent is also useful as a therapeutic agent for corneal scarring and conjunctival scarring both associated with keratoconjunctive disorders.

TECHNICAL FIELD

The present invention relates to a therapeutic agent for akeratoconjunctive disorder containing a RARγ agonist as an effectiveingredient.

BACKGROUND ART

The cornea is a transparent avascular tissue with a diameter of about 1cm, covering the front surface of an eye ball. The conjunctiva is amucous membrane covering the back surface of the eye lid and the surfaceof an eye ball which is posterior to the corneal limbus. The cornea andthe conjunctiva serve an important role in vision. It is known thatvisual function is seriously affected when a disorder occurs therein.Keratoconjunctive disorders induced by various diseases such as cornealulcer, keratitis, and dry eye are disorders caused by a delay inrecovery from a disorder due to some reason such as an external injuryor by a disorder that has become chronic. Since the cornea is a tissuethat is connected to the conjunctiva, such diseases negatively affecteach other in the normal construction of the epithelium and in somecases harm the structure or functioning of the corneal stroma orendothelium.

Collagen (especially type I collagen) is known as one of therepresentative stromal components of corneal parenchymal tissue. Afunctional disorder resulting from stromal degradation occurs indiseases due to a keratoconjunctive disorder. Thus, suppression ofdegradation of collagen (especially type I collagen) is consideredeffective against diseases caused by a keratoconjunctive disorder.

In a keratoconjunctive disorder, scar tissue, which is often formedafter an inflammation has calmed, may obstruct the visual function. Forthis reason, suppression of collagen contraction, if possible, isconsidered effective against contraction and formation of scars(hereinafter, referred together as “scar formation”) as in theabove-described suppression of collagen degradation.

Patent Literature 1 describes that all-trans retinoic acid (hereinafter,also referred to as ATRA) promotes corneal regeneration. However, theeffect thereof is weak and the detailed mechanism thereof is yet to beelucidated.

Further, ATRA is an agonist of retinoic acid receptors (hereinafter,also referred to as RAR). However, since ATRA does not have selectivitywith respect to RAR subtypes RARα, RARβ, and RARγ, the contribution ofeach RAR subtype to corneal regeneration effects is unknown.

Meanwhile, RAR is involved in various effects such as growth,morphogenesis and differentiation in many cells, such as inflammatorycells, immune cells, and structural cells. Further, it is verified thatthere is a difference in the distribution of RAR subtypes depending onthe tissue or organ of a mammal.

Some of the effects of RAR are undesirable, such as increase intriglyceride due to RARα. Thus, the specificity or selectivity withrespect to subtypes in compounds with RAR agonist activity is expectedto lead to reduction in risk of side effects.

For the above reasons, there is a demand for RAR agonists, which have astrong effect of suppressing keratoconjunctive disorders and are highlysafe based on subtype selectivity.

Patent Literatures 2 and 3 disclose the RAR agonist(E)-4-(2-{3-[(1H-pyrazole-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-yl}vinyl)benzoic acid and the derivatives thereof. Further, Patent Literature2 describes that(E)-4-(2-{3-[(1H-pyrazole-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-yl}vinyl)benzoic acid is useful against pulmonary emphysema, cancer, anddermatosis. Patent Literature 3 describes that the above-describedagonist is useful against neurological pain.

In addition, Non-Patent Literature 1 describes that the RAR agonist6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene acid induces apoptosisin lung cancer cells.

Furthermore, Patent Literature 4 describes that the RAR agonist3-fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-yl)acetylamino]benzoicacid is useful in muscle repair or regeneration.

However, pharmacological effects on keratoconjunctive disorders or scarformation resulting from keratoconjunctive disorders are not discussedor reported for any of the RAR agonists. In addition, there is noliterature that suggests such an effect.

CITATION LIST Patent Literature

-   [PTL 1] Japanese Laid-Open Publication No. 2009-235031-   [PTL 2] International Publication No. WO 2002/028810-   [PTL 3] International Publication No. WO 2008/057930-   [PTL 4] Japanese Laid-Open Publication No. 2013-536855

Non Patent Literature

-   [NPL 1] Sun S Y et al., Cancer Research 62(8): 2430-2436 (2002)

SUMMARY OF INVENTION Technical Problem

The search for a drug that is effective against ophthalmic diseases,especially keratoconjunctive disorders, is an objective that isimportant and of interest. The objective of the present invention is toprovide a therapeutic agent that has an effect of suppressing akeratoconjunctive disorder and is highly safe based on subtypeselectivity.

Solution to Problem

After diligent research to find a drug that is effective againstkeratoconjunctive disorders, the inventors discovered that the RARγagonist(E)-4-(2-{3-[(1H-pyrazole-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-yl}vinyl)benzoic acid (R667: hereinafter, also referred to as “RARγ agonistA”) exerts an excellent effect of ameliorating keratoconjunctivedisorders as well as scar formation associated with keratoconjunctivedisorders by pharmacological tests using rabbit keratocytes andsubconjunctival fibroblasts, wherein a potent effect of suppressingcollagen degradation as well as a significant effect of suppressingcollagen contraction were demonstrated. Furthermore, it was discoveredthat other RARγ agonists,6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene acid (CD437:hereinafter, also referred to as “RARγ agonist B”) and3-fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-yl)acetylamino]benzoicacid (BMS961: hereinafter, also referred to as “RARγ agonist C”) alsoexhibit a significant effect of suppressing collagen degradation inpharmacological tests using rabbit keratocytes to complete the presentinvention.

Specifically, the present invention is [1] a therapeutic agent for akeratoconjunctive disorder, comprising a RARγ agonist as an effectiveingredient, [2] the therapeutic agent of the above-described [1],wherein the RARγ agonist is(E)-4-(2-{3-[(1H-pyrazole-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-yl}vinyl)benzoic acid, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene acid,3-fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-yl)acetylamino]benzoicacid, an ester thereof, or a salt thereof, [3] the therapeutic agent ofabove-described [1] or [2], wherein the keratoconjunctive disorder isselected from the group consisting of corneal ulcer, corneal epithelialabrasion, keratitis, dry eye, conjunctivitis, chronic superficialkeratitis, corneal erosion, persistent corneal disorders, superficialpunctate keratopathy, corneal epithelial defects, conjunctivalepithelial defects, keratoconjunctivitis sicca, superior limbickeratoconjunctivitis, filamentary keratoconjunctivitis, infectiouskeratitis, noninfectious keratitis, infectious conjunctivitis,noninfectious conjunctivitis, corneal cicatrization, and conjunctivalcicatrization, [4] the therapeutic agent according to any one of theabove-described [1]-[3], wherein a form of administration isinstillative administration or oral administration, and [5] thetherapeutic agent accordingly to any one of the above-described [1]-[4],wherein a dosage form is an instillation, an ophthalmic ointment, aninjection, a tablet, a granule, a fine granule, a powder or a capsule.

Advantageous Effects of Invention

RARγ agonists, which are effective ingredients of the therapeutic agentfor a keratoconjunctive disorder of the present invention, are useful asa therapeutic agent for keratoconjunctive disorders, such as cornealulcer, corneal epithelial abrasion, keratitis, dry eye, conjunctivitis,chronic superficial keratitis, corneal erosion, persistent cornealdisorders, superficial punctate keratopathy, corneal epithelial defects,conjunctival epithelial defects, keratoconjunctivitis sicca, superiorlimbic keratoconjunctivitis, filamentary keratoconjunctivitis,infectious keratitis, noninfectious keratitis, infectious conjunctivitisor noninfectious conjunctivitis, by strongly suppressingkeratoconjunctive collagen degradation.

Further, RARγ agonists, which are effective ingredients of thetherapeutic agent for a keratoconjunctive disorder of the presentinvention, are also useful as a therapeutic agent for cornealcicatrization or conjunctival cicatrization associated with akeratoconjunctive disorder by strongly suppressing keratoconjunctivecollagen contraction.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the relationship between the concentration(nM) of RARy agonist A (R667) and collagen degradation (amount (μg) ofhydroxyproline per well). The vertical axis represents the numericalvalue (%) when the amount of hydroxyproline of the control is set to100.

FIG. 2 is a graph showing the relationship between the concentration(nM) of RARγ agonist A (R667) and collagen contraction (diameter (mm) ofcollagen gel in the dish) when using subconjunctival fibroblasts,wherein

indicates the presence of a statistically significant difference(p<0.05).

FIG. 3 is a graph showing the relationship between the concentration(μM) of RARγ agonist A (R667) and collagen contraction (diameter (mm) ofcollagen gel in the dish) when using keratocytes, wherein

indicates the presence of a statistically significant difference(p<0.05).

FIG. 4 is a graph showing the relationship of the concentrations (nM) ofRARγ agonist A (R667), RARγ agonist B (CD437), and RARγ agonist C(BMS961) with respect to collagen degradation, when using keratocytes.The vertical axis represents the ratio (Ratio) of the amount (μg) ofhydroxyproline per well when a RARγ agonist and a stimulant are added,in cases where the amount (μg) of hydroxyproline per well when a RARγagonist and a stimulant are not added is set to 1, wherein

indicates the presence of a statistically significant difference(p<0.05).

FIGS. 5A and 5B show the relationship between the concentration (μM) ofRARγ agonist A (R667) and the expression and activation of matrixmetalloproteinases (MMP) when using keratocytes. The top row of FIG. 5Ashows the expression and activation of MMP-1, the bottom row of FIG. 5Ashows the expression and activation of MMP-3, and FIG. 5B shows theexpression and activation of MMP-2 and MMP-9.

FIGS. 6A and 6B show the results of observation when alipopolysaccharide (LPS) and a RARγ agonist A (R667) are administeredinto the corneal stroma in one eye of male Japanese white rabbits. Thetop row (FIG. 6A) (Vehicle) is the result of administering only asolution that does not contain the RARγ agonist A (R667) of the presentinvention, and the bottom row (FIG. 6B) (0.1% R667) is the result ofadministering a solution containing RARγ agonist A (R667).

DESCRIPTION OF EMBODIMENTS

The therapeutic agent for a keratoconjunctive disorder of the presentinvention is not particularly limited and may be any therapeutic agenthaving a RARγ agonist as the effective ingredient. The RARγ agonist ofthe present invention refers to a compound that can promote theactivation of a RARγ receptor by binding to a RARγ receptorsignificantly in comparison to RAR α receptors or RAR β receptors.

Examples of such RARγ agonists include(E)-4-(2-{3-[(1H-pyrazole-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-yl}vinyl)benzoic acid (R667) represented by the following formula (I),6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene acid (CD437)represented by the following formula (II),3-fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-yl)acetylamino]benzoicacid (BMS961) represented by the following formula (III),(2E)-3-(4-carboxyphenyl)-1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-yl)-2-propene-1-oneoxime (NRX204647: hereinafter, also referred to as “RARγ agonist D”)represented by the following formula (IV),4-[7-(1-adamantly)-6-hydroxynaphthalene-2-yl] benzoic acid (CD1530:hereinafter, also referred to as “RARγ agonist E”) represented by thefollowing formula (V), esters of such compounds (RARγ agonists A, B, C,D, and E), and salts of such compounds (RARγ agonists A, B, C, D, andE). Preferred examples of RARγ agonists include RARγ agonist A, RARγagonist B, RARγ agonist C, esters of such compounds (RARγ agonists A, B,and C), and salts of such compounds (RARγ agonists A, B, and C).

Further, other embodiments of the present invention include a method oftreating a keratoconjunctive disorder characterized by administering theRARγ agonist of the present invention to a subject, a RARγ agonist ofthe present invention for use as a therapeutic agent for akeratoconjunctive disorder, and use of the RARγ agonist of the presentinvention in the preparation of a therapeutic agent for akeratoconjunctive disorder.

RARγ agonist A, which is one of the effective ingredients of thetherapeutic agent for a keratoconjunctive disorder of the presentinvention, an ester thereof and a salt thereof are known compoundsdescribed in Patent Literature 2. They can be manufactured in accordancewith the method described in Patent Literature 2 or purchased as acommercially-available product. Examples of the commercially-availableproducts include product name: palovarotene manufactured by ShanghaiHaoyuan Chemexpress.

Among the effective ingredients of the therapeutic agents for akeratoconjunctive disorder of the present invention, RARγ agonist B is aknown compound described in Non-Patent Literature 1 and PatentLiterature 4, RARγ agonist C is a known compound described in PatentLiterature 4, and RARγ agonists D and E are known compounds described inthe following document (Shimono K. et al., Nat Med. 17(4): 454-460(2011)). Said compounds, esters thereof and salts thereof can bemanufactured in accordance with a conventional method or purchased as acommercially-available product. Examples of commercially availableproducts include product name: CD437 (ab141305) manufactured by Abcamand product name: CD437 manufactured by Tocris Bioscience for RARγagonist B, product name: BMS961 manufactured by Tocris Bioscience forRARγ agonist C, and product name: CD1530 manufactured by Santa Cruzbiotechnology and product name: CD1530 manufactured by Tocris Biosciencefor RARγ agonist E.

Esters in the aforementioned esters of RARγ agonists A, B, C, D, and E,which are effective ingredients of the therapeutic agent for akeratoconjunctive disorder of the present invention, are notparticularly limited and may be any ester converted to RARγ agonists A,B, C, D, or E in a reaction by an enzyme or the like under physiologicalconditions in vivo. Such esters include: esters generated by reactionwith a primary alcohol, such as methanol, ethanol, propanol, hexanol, ordodecanol; esters generated by reaction with a secondary alcohol such asisopropanol, s-butanol, or 1-ethylpropanol; esters generated by reactionwith a tertiary alcohol such as t-butanol or 1-methyl-1-ethylpropanol;and esters generated by reaction with an amino alcohol such as2-aminoethanol.

The above-described esters can be manufactured by a known method fromthe aforementioned RARγ agonists A, B, C, D, E, or a syntheticintermediate thereof.

Salts in the aforementioned salts of RARγ agonists A, B, C, D, and E,which are effective ingredients of the therapeutic agent for akeratoconjunctive disorder of the present invention, are notparticularly limited and may be any pharmaceutically acceptable salts.Such salts include (1) as an acid addition salt, inorganic acid saltssuch as hydrochloride, hydrobromic acid salt, hydroiodic acid salt,nitric acid salt, sulfuric acid salt, and phosphoric acid salt; andorganic acid salts such as acetic acid salt, trifluoroacetic acid salt,benzoic acid salt, oxalic acid salt, malonic acid salt, succinic acidsalt, maleic acid salt, fumaric acid salt, tartaric acid salt, citricacid salt, methanesulfonic acid salt, ethanesulfonic acid salt,trifluoromethanesulfonic acid salt, benzenesulfonic acid salt,p-toluenesulfonic acid salt, glutamic acid salt, and aspartic acid saltand (2) as a basic salt, metal salts such as sodium salt, potassiumsalt, calcium salt and magnesium salt; inorganic salts such as ammoniumsalt; and organic amine salts such as triethylamine salt and guanidinesalt.

In the present invention, a keratoconjunctive disorder refers to acondition where a cornea or a conjunctiva is damaged due to variousfactors such as an abnormality in lacrimal fluid, metabolic abnormality,or external injury. Examples thereof include corneal ulcer, cornealepithelial abrasion, keratitis, dry eye, conjunctivitis, chronicsuperficial keratitis, corneal erosion, persistent corneal disorders,superficial punctate keratopathy, corneal epithelial defects,conjunctival epithelial defects, keratoconjunctivitis sicca, superiorlimbic keratoconjunctivitis, filamentary keratoconjunctivitis,infectious keratitis, noninfectious keratitis, infectious conjunctivitisand noninfectious conjunctivitis. Suppressing action on collagendegradation exerts an excellent ameliorating effect. Further, in thepresent invention, corneal cicatrization (scar formation on the cornea)and conjunctival cicatrization (scar formation on the conjunctiva)associated with a keratoconjunctive disorder are also examples ofkeratoconjunctive disorders. Suppressing action on collagen contractionexerts an excellent ameliorating effect.

The therapeutic agent for a keratoconjunctive disorder of the presentinvention can be administered orally or parenterally (intravenousadministration, intramuscular administration, intraperitonealadministration, percutaneous administration, intratrachealadministration, intracutaneous administration, or subcutaneousadministration) in a form of a tablet, capsule, powder, syrup, granule,fine granule, pill, liquid formulation, suspension, emulsion,percutaneous absorption agent, suppository, ointment (preferablyophthalmic ointment), lotion, inhalant, or injection manufactured bymixing in a suitable pharmacologically acceptable additive.

These formulations are manufactured by a well-known method by using anadditive such as an excipient, lubricant, binding agent, disintegrator,emulsifier, stabilizer, flavoring agent or diluent.

Examples of excipients include organic excipients and inorganicexcipients. Examples of organic excipients include: sugar derivativessuch as lactose, sucrose, glucose, mannitol, and sorbitol; starchderivatives such as corn starch, potato starch, α-starch and dextrin;cellulose derivatives such as crystalline cellulose; gum arabic;dextran; and pullulan. Examples of inorganic excipients include: lightanhydrous silicic acid; and sulfuric acid salts such as calcium sulfate.

Examples of lubricants include: stearic acid; metal salts of stearicacid such as calcium stearate and magnesium stearate; talc; colloidalsilica; wax such as beeswax and spermaceti; boric acid; adipic acid;sulfuric acid salts such as sodium sulfate; glycol; fumaric acid; sodiumbenzoate; D,L-Leucine, sodium lauryl sulfate; silicic acids such assilica and silicic acid hydrate; and the starch derivatives for theabove-described excipients.

Examples of binding agents include hydroxypropyl cellulose,hydroxypropyl methylcellulose, polyvinylpyrrolidone, macrogol and thecompounds described above shown for excipients.

Examples of disintegrators include: cellulose derivatives such ashydroxypropyl cellulose with a low degree of substitutions,carboxymethyl cellulose, calcium carboxymethyl cellulose, and internallycrosslinked calcium carboxymethyl cellulose; crosslinkedpolyvinylpyrrolidone; and chemically modified starch or cellulosederivatives such as carboxymethyl starch and sodium carboxymethylstarch.

Examples of emulsifiers include: colloidal clay such as bentonite andveegum; anionic surfactants such as sodium lauryl sulfate; cationicsurfactants such as benzalkonium chloride; and non-ionic surfactantssuch as polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acidester, and sucrose fatty acid ester.

Examples of stabilizers include: para-hydroxybenzoic acid esters such asmethylparaben and propylparaben; alcohols such as chlorobutanol, benzylalcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such asphenol and cresol; thimerosal; acetic anhydride; and sorbic acid.

Examples of flavoring agents include: sweeteners such as sodiumsaccharin and aspartame; acidulants such as citric acid, malic acid, andtartaric acid; and flavors such as lemon extract and orange extract.

Diluents are generally compounds used as a diluent. Examples thereofinclude lactose, mannitol, glucose, sucrose, calcium sulfate,hydroxypropyl cellulose, microcrystalline cellulose, water, ethanol,polyethylene glycol, propylene glycol, glycerin, starch,polyvinylpyrrolidone, and mixtures thereof.

The therapeutic agent for a keratoconjunctive disorder of the presentinvention includes those in a form of instillation in addition to theabove-described dosage forms. The agent can be formulated with awell-known method by suitably blending in an isotonizing agent, buffer,pH regulator, solubilizer, thickener, stabilizer, preservative(antiseptic) or the like as an additive. Further, it is also possible toobtain a stable instillation by adding a pH regulator, thickener,dispersant or the like to prepare suspension of a drug.

Examples of isotonizing agents include glycerin, propylene glycol,sodium chloride, potassium chloride, sorbitol and mannitol.

Examples of buffers include phosphoric acid, phosphate, citric acid,acetic acid, and ε-aminocaproic acid.

Examples of pH regulators include hydrochloric acid, citric acid,phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide,boric acid, borax, disodium hydrogen phosphate, sodium dihydrogenphosphate, sodium carbonate, and sodium bicarbonate.

Examples of solubilizers include polysorbate 80, polyoxylethylenehydrogenated castor oil 60, and macrogol 4000.

Examples of thickeners and dispersants include: cellulose polymers suchas hydroxypropyl methylcellulose and hydroxypropyl cellulose; polyvinylalcohols; and polyvinylpyrrolidone. Further, examples of stabilizersinclude edetic acid and sodium edetate.

Examples of preservatives (antiseptics) include commonly-used sorbicacid, potassium sorbate, benzalkonium chloride, benzethonium chloride,methyl parahydroxybenzoate, propyl parahydroxybenzoate, andchlorobutanol. It is also possible to use these preservatives incombination.

An instillation may have any pH within a range acceptable for anophthalmic formulation, but the pH is desirably set to 4.0-8.5.

For ointments (preferably ophthalmic ointments), a commonly-used basesuch as white petrolatum or liquid paraffin can be used for preparation.

The dosage of the therapeutic agent for a keratoconjunctive disorder ofthe present invention can be appropriately changed in accordance withthe dosage form, severity of symptoms of a patient to whom the agent isto be administered, age, weight, judgment of a physician or the like.For oral agents, it is generally possible to administer 0.01-5000 mg,preferably 0.1-2500 mg, and more preferably 0.5-1000 mg per day for anadult in one or several doses. For instillations, it is possible toadminister those with an effective ingredient concentration of0.000001-10% (W/V), preferably 0.00001-3% (W/V), and more preferably0.0001-1% (W/V), in one or several daily doses. For ophthalmicointments, it is possible to administer those with an effectiveingredient concentration of 0.00001-10% (W/W), preferably 0.0001-3%(W/V), and more preferably 0.001-1% (W/W), in one or several dailydoses.

Hereinafter, the present invention is explained in further detail whileproviding Examples (Test Examples and Formulation Examples). However,the scope of the present invention is not limited thereto.

Example 1 (Test Example) Test on Suppression of Three-DimensionalCollagen Gel Degradation in Normal Rabbit Primary Keratocytes by RARγAgonist A

Normal rabbit corneal cells were used to assess the suppression effectof a tested compound on three-dimensional collagen gel degradation inaccordance with the method of Nishida et al (Investigative Ophthalmology& Visual Science 42: 1247-1253 (2001)).

Primary keratocytes collected from a normal rabbit eye ball was grown toa confluent state and detached from a culture slide with 0.05%Trypsin-EDTA. After washing in a serum free medium (product number11095; Gibco), the number of cells were counted. The obtained primarykeratocytes were mixed with the type I collagen solution Cellmatrix TypeI-A (product number 637-00653; Nitta Gelatin Inc.) and ReconstitutionBuffer (product number 635-00791; Nitta Gelatin Inc.) and dispensed intoa 24-well plate so that the final concentration would be 1×10⁵cells/well to make a collagen gel.

After making the gel, culture was started (under conditions of 37° C.and 5% CO₂) by adding to a MEM medium a RARγ agonist A (R667)-containingdimethyl sulfoxide solution (R667 concentration: 0.1 nM, 1 nM, 10 nM,100 nM, or 1000 nM) or a RARγ agonist-free dimethyl sulfoxide solutionas a control and 10 ng (final concentration 10 ng/ml) of IL-113 (productnumber 201-LB-005; R&D Systems) and 60 μg (final concentration 60 μg/ml)of Plasminogen (product number P9156; Sigma) as a stimulant, andoverlaying the medium on the previously prepared collagen gel.

After 48 hours of culturing, the supernatant was ultrafiltrated, 100 μlof concentrated hydrochloric acid was added and heated, and the collagenwas hydrolyzed. The reaction solution after the hydrolysis was driedunder a nitrogen gas atmosphere by using a Dry Thermo Unit (DTU-2C,Taitec Co. Ltd.) and an evaporation head (E1-20 Taitec Co. Ltd.) andthen dissolved in 500 μl of ultrapure water. The amount ofhydroxyproline, which is the collagen degradation product in thesolution, was measured in accordance with the method of Bergman et al(Analytical Chemistry 35(12): 1961-1965 (1963)) to assess the effect ofsuppressing collagen degradation of RARγ agonist A. The results areshown in FIG. 1.

In the present test, RARγ agonist A exhibited a dosage-dependent effectof suppressing keratoconjunctive collagen degradation.

Formulation Example (Drug Formulation Example 1) Instillation

In 100 ml RARγ agonist A 100 mg Sodium chloride 800 mg Polysorbate 80appropriate amount Disodium hydrogen phosphate appropriate amount Sodiumdihydrogen phosphate appropriate amount Sterile purified waterappropriate amount

RARγ agonist A and the other components described above are added tosterile purified water. The solution is thoroughly mixed to prepare aninstillation. It is possible to prepare an instillation with aconcentration of 0.05% (W/V), 0.3% (W/V), 0.5% (W/V), or 1% (W/V) bychanging the amount of RARγ agonist A or the like that is added.

(Drug Formulation Example 2) Ophthalmic Ointment

In 100 g RARγ agonist A 0.3 g Liquid paraffin 10.0 g  White petrolatumappropriate amount

RARγ agonist A is added to homogeneously-melted white petrolatum andliquid paraffin. The mixture is thoroughly mixed and then graduallycooled to prepare an ophthalmic ointment. It is possible to prepare anophthalmic ointment with a concentration of 0.05% (W/W), 0.1% (W/W),0.5% (W/W), or 1% (W/W) by changing the amount of RARγ agonist A or thelike that is added.

(Drug Formulation Example 3) Tablet

In 100 mg RARγ agonist A 1 mg Lactose 66.4 mg   Corn starch 20 mg Calcium carboxymethyl cellulose 6 mg Hydroxypropyl cellulose 6 mgMagnesium stearate 0.6 mg  

RARγ agonist A, corn starch and lactose are mixed in a mixer. Calciumcarboxymethyl cellulose and hydroxypropyl cellulose are added to themixture for granulation. The particle size of the resulting granules isadjusted after drying. Magnesium stearate is added to and mixed with theadjusted granules and the mixture is made into tablets with a tabletingmachine. Further, it is possible to prepare tablets with the content of0.1 mg, 10 mg, or 50 mg in 100 mg by changing the amount of RARγ agonistA or the like that is added.

Example 2 (Test Example) Test on Suppression of Three-DimensionalCollagen Gel Contraction in Various Primary Cells Derived from NormalRabbits by RARγ Agonist A

Primary subconjunctival fibroblasts and primary keratocytes were used toassess the suppression effect of a tested compound on three-dimensionalcollagen gel contraction in accordance with the method of Nishida et al.

As in Example 1, primary subconjunctival fibroblasts were grown anddetached from a culture slide. After washing, a cell suspension wasmade. The resulting suspension (1.1×10⁷ cells/well MEM), type I collagensolution (5 mg/ml), 10×MEM, Reconstitution Buffer, and water were mixedon ice at 0.2:7:1:1:1.8 (volume ratio). A culture dish coated with 1%BSA was inoculated with 0.5 ml of the mixture, which was incubated forone hour at 37° C. to make a collagen gel.

Then, 0.5 ml of serum free media, to which a certain amount (1 ng/ml) ofTGF-β1 (R&D Systems) and RARγ agonist A at each of the concentrations 1nM, 10 nM, and 100 nM were added, were each added onto theabove-described gel. The gel, together with a gel to which an agent-freeserum-free medium was added, was continuously incubated at 37° C. Thediameter of gels was measured from the time when 24 hours has past. Theresults of measuring the diameters of the gels after 48 hours are shownin FIG. 2.

The gel diameter was measured for primary keratocytes in a similarmanner to the primary subconjunctival fibroblasts. The results are shownin FIG. 3.

It can be seen from FIGS. 2 and 3 that RARγ agonist A not only cansuppress collagen degradation, but also collagen gel contraction due toTGF using subconjunctival fibroblasts or keratocytes. This demonstratesthat RARγ agonist A contributes to collagen turn over and has an effectof suppressing tissue remodeling that occurs after inflammation,hemorrhage, infection, surgery, or injury in an ophthalmic tissue, i.e.,fibrillation or cicatrization.

Example 3 (Test Example) Test on Suppression of Three-DimensionalCollagen Gel Degradation in Primary Keratocytes Derived from NormalRabbits by RARγ Agonist A, B, or C

Primary keratocytes were used to assess the suppression effect of atested compound on three-dimensional collagen gel degradation by thesame method as in Example 1 in accordance with the method of Nishida etal.

As in Example 1, primary keratocytes were grown and detached from aculture slide. After washing, a cell suspension was made. The resultingsuspension (1.1×10⁷ cells/well MEM), type I collagen solution (5 mg/ml),10×MEM, Reconstitution Buffer, and water were mixed on ice at0.2:7:1:1:1.8 (volume ratio). A culture dish coated with 1% BSA wasinoculated with 0.5 ml of the mixture, which was incubated for one hourat 37° C. to make a collagen gel.

After making the gel, culture was started (under conditions of 37° C.and 5% CO₂) by adding to a MEM medium a RARγ agonist A (R667)-containingdimethyl sulfoxide solution (1 nM), RARγ agonist B (CD437)-containingdimethyl sulfoxide solution (1 nM, 10 nM), RARγ agonist C(BMS961)-containing dimethyl sulfoxide solution (10 nM), or a RARγagonist-free dimethyl sulfoxide solution as a control, and 10 ng (finalconcentration 10 ng/ml) of IL-1β (product number 201-LB-005; R&DSystems) and 60 μg (final concentration 60 μg/ml) of Plasminogen(product number P9156, Sigma Aldrich) as a stimulant, and overlaying themedium on the previously prepared collagen gel.

After 48 hours of culturing, collagen was hydrolyzed and the amount ofhydroxyproline, which is the collagen degradation product, was measuredby the same method as Example 1 to assess the effect of suppressingcollagen degradation of each RARγ agonist. The results are shown in FIG.4.

In the present test, not only RARγ agonist A, but also RARγ agonists Band C exhibited an effect of suppressing keratoconjunctive collagendegradation.

Example 4 (Test Example) Test on Suppression of the Expression andActivation of MMP-1, 2, 3, and 9 by RARγ Agonist A

It is believed that secretion or expression of protease, i.e., matrixmetalloproteinases (MMP), is associated with the degradation of type Icollagen. In this regard, suppression of expression and activation ofMMP-1, 2, 3, and 9 by RARγ agonist A was investigated.

Primary keratocytes collected from a normal rabbit eye ball werecultured for 24 hours in a serum-free MEM medium. RARγ agonist A (R667)(R667 concentration: 1×10⁻⁶ μM, 1×10⁻⁵ μM, 1×10⁻⁴ μM, 1×10⁻³ μM, 1×10⁻²μM) was added to the obtained culture solution and 12 hours ofpre-treatment was performed. As a positive control, 10 nM ofdexamethasone (Dex), which is a synthetic steroid, was added andpre-treatment was similarly performed. Then, IL-113 (0.1 ng/ml) wasadded for stimulation, and the supernatant was collected after 24 hours.The following western blot analysis and gelatin zymography analysis wereused on the collected culture solution.

(Western Blot Analysis)

After developing the supernatant of the collected culture solution inSDS-PAGE using 10% polyacrylamide gel, the separated proteins weretransferred onto a nitrocellulose filter. Then, non-specific sites onthe nitrocellulose filter were blocked and incubated for 24 hours at 4°C. with anti-human MMP-1 antibodies (R&D Systems) and anti-rabbit MMP-3antibodies (Daiichi Fine Chemical Co., Ltd). ECL® reagent (GEHealthcare) was used for detection.

The results are shown in FIG. 5A. The band for each of the non-activePro-MMP-1, Pro-MMP-3 and active MMP-1, MMP-3 was diminished, where theextent thereof was dependent on the concentration of RARγ agonist A.Thus, it was revealed that RARγ agonist A concentration-dependentlysuppresses the expression and activation of MMP-1 and MMP-3.

(Gelatin Zymography Analysis)

After developing the supernatant of the collected culture solution onSDS-PAGE using 10% polyacrylamide gel containing 0.1% of gelatin, it wasincubated for one hour at room temperature with a TBS solutioncontaining 2.5% Triton X-100. The gel after incubation was dyed with asolution of Coomassie Brilliant Blue (Wako Pure Chemical Industries,Ltd.) and decolored with a 5% methanol-7.5% acetic acid solution(Nacalai Tesque, Inc).

The results are shown in FIG. 5B. The band for each of the non-activePro-MMP-2, Pro-MMP-9 and active MMP-2, MMP-9 was diminished, where theextent thereof was dependent on the concentration of RARγ agonist A.Thus, it was revealed that RARγ agonist A concentration-dependentlysuppresses the expression and activation of MMP-2 and MMP-9.

Example 5 (Test Example) Test on Suppression of Corneal Opacity andUlceration with RARγ Agonist A

Systemic anesthesia was applied to male Japanese white rabbits (bodyweight 2.5-3.5 kg, 27 rabbits) by intramuscular administration of asolution of a mixture of ketamine and xylazine. Then, topical anesthesiawas applied by a 0.4% oxybuprocaine hydrochloride instillation.Furthermore, 30 μl of 1% LPS (Sigma Aldrich) was injected into thecorneal stroma of one eye, but not in the other eye.

50 μl of 0.1% PBS/0.1% polysorbate 80 solution containing RARγ agonist A(R667: 0.1%, 24 mM) was instillatively administered to the rabbitsinjected with LPS twice after the injection on the day of LPS injectionand four times daily thereafter until day 10 by using a micropipette. Asa control, a 0.1% PBS/0.1% polysorbate 80 solution free of RARγ agonistA (Vehicle) was similarly administered. FIGS. 6A and 6B showrepresentative examples of a control and a case where RARγ agonist A wasadministered.

In FIGS. 6A and 6B, corneal opacity and ulceration were observed in thecontrol (Vehicle) shown in the top row (FIG. 6A). However, in a casewhere RARγ agonist A was administered (0.1% R667) shown in the bottomrow (FIG. 6B), corneal opacity and ulceration were not observed. Thus,it was revealed that RARγ agonist A suppresses corneal opacity andulceration.

INDUSTRIAL APPLICABILITY

RARγ agonists, which are effective ingredients of the therapeutic agentfor a keratoconjunctive disorder of the present invention, are useful inpreventing or as a therapeutic agent for keratoconjunctive disorders,such as corneal ulcer, corneal epithelial abrasion, keratitis, dry eye,conjunctivitis, chronic superficial keratitis, corneal erosion,persistent corneal disorders, superficial punctate keratopathy, cornealepithelial defects, conjunctival epithelial defects,keratoconjunctivitis sicca, superior limbic keratoconjunctivitis,filamentary keratoconjunctivitis, infectious keratitis, noninfectiouskeratitis, infectious conjunctivitis and noninfectious conjunctivitis,by strongly suppressing collagen degradation. Further, said RARγagonists are also useful as a therapeutic agent for cornealcicatrization or conjunctival cicatrization associated with akeratoconjunctive disorder by strongly suppressing collagen contraction.

1-5. (canceled)
 6. A method of preventing or treating conjunctivitis ina subject in need thereof, comprising administering to the subject(E)-4-(2-{3-[(1H-pyrazole-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-yl}vinyl)benzoicacid (R667), or an ester or salt thereof, in an amount effective toprevent or treat the conjunctivitis.
 7. The method of claim 6, whereincorneal damage or scarring is inhibited.
 8. The method of claim 6,wherein the R667, or a salt thereof, is in the form of a powder, syrup,granule, liquid formulation, suspension, emulsion, ointment, orinstillation.
 9. The method of claim 8, wherein the ointment is anophthalmic ointment.
 10. The method of claim 9, wherein the ointmentcomprises a solubilizer.
 11. The method of claim 10, wherein thesolubilizer is selected from the group consisting of polysorbate 80,polyoxylethylene hydrogenated castor oil 60, and macrogol
 4000. 12. Themethod of claim 11, wherein the solubilizer is polysorbate
 80. 13. Themethod of claim 9, wherein the concentration of the R667, or a saltthereof, in the ophthalmic ointment administered to the subject is from0.0001% to 3% (w/v).
 14. The method of claim 13, wherein theconcentration of the R667, or a salt thereof, in the ophthalmic ointmentadministered to the subject is from 0.001% to 1% (w/v).
 15. The methodof claim 8, wherein the R667, or a salt thereof, is in the form of aninstillation.
 16. The method of claim 15, wherein the instillationcomprises a solubilizer.
 17. The method of claim 16, wherein thesolubilizer is selected from the group consisting of polysorbate 80,polyoxylethylene hydrogenated castor oil 60, and macrogol
 4000. 18. Themethod of claim 17, wherein the solubilizer is polysorbate
 80. 19. Themethod of claim 15, wherein the concentration of the R667, or a saltthereof, in the instillation administered to the subject is from0.00001% to 3% (w/v).
 20. The method of claim 19, wherein theconcentration of the R667, or a salt thereof, in the instillationadministered to the subject is from 0.0001% to 1% (w/v).
 21. The methodof claim 20, wherein the concentration of the R667, or a salt thereof,in the instillation administered to the subject is 0.3% (w/v), 0.5%(W/V), or 1% (W/V).
 22. The method of claim 6, wherein the administeringis administering by instillation.
 23. The method of claim 6, wherein theR667, or a salt thereof, is administered to the subject in one or moredaily doses.